System and Method for the Production, Formulation and Use of Conditioned Media, Cultured Cells and the Factors Included Therein

ABSTRACT

A technology regarding the production, formulation and use of conditioned media and the factors included therein is disclosed. The conditioned media may be inoculated with animal cells, plant cells and any combination thereof. The inoculations may occur simultaneous or at different times. Cells retrieved from different areas of the animal and/or the plant may also be cultured together to form conditioned media and associated growth factors.

RELATIONSHIPS TO PRIOR APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/702,825, filed Jul. 24, 2018, the entire contents of which are hereby fully incorporated herein by reference for all purposes.

COPYRIGHT STATEMENT

This patent document contains material subject to copyright protection. The copyright owner has no objection to the reproduction of this patent document or any related materials in the files of the United States Patent and Trademark Office, but otherwise reserves all copyright whatsoever.

FIELD OF THE INVENTION

This invention relates to conditioned media and factors generated and included therein. More particularly, this invention relates to specifically designed conditioned media and factors, their formulation, their method of production and their methods and areas of use.

BACKGROUND

Most methodologies have been developed to maintain cells in culture. In parallel, several culture media with different composition have been formulated to culture and expand different cell types in vitro. Once the cells are isolated from their original niche, they start losing signals from their natural environment and change their phenotype. In order to maintain the original phenotype, complex culture media have been developed. However, some cells still lose some differentiation characteristics.

When the cells are cultured in an adequate environment including soluble nutrients, gases, temperature and humidity, they are stimulated for proliferation. In these conditions, the cells produce extracellular matrix components that allow the cells to attach to the culture dish and establish physical contact with their neighboring cells. The cell-cell contact is important for cell-cell communication. However, there is another type of cell-cell communication through soluble factors that the cells release to communicate to other cells. These chemical communications have great impact on cellular behavior and function.

In vivo, the clearest example occurs during wound healing. When the skin is damaged, the epithelial cells in the epidermis and other cells such as fibroblasts and endothelial cells in the dermis respond immediately by releasing soluble factors to the closer environment and to the blood stream. These factors also provide signals to neighboring cells for further activation and differentiation.

In vitro, a similar process can take place, but the cell-cell interactions are limited to the culture dish area. Also, the factors released by cells grown in vitro are able to interact with specific membrane receptors and induce cell activation, migration, apoptosis, or differentiation. The in vitro system allows the detailed study of all these factors. Since these factors are released in the media where the cells are growing, it is possible to collect this media and analyze the profiles and levels of factors that the media contains.

It is also possible to manipulate the content of these media with different patterns of signals that can be introduced to the cells in culture. For example, vascular endothelial growth supplement (VEGS) is normally added to the media when endothelial cells are isolated for the first time (primary cultures). At present, the conditioned media derived from different cell lines has been analyzed and several factors including but not limited to, cytokines and chemokines, have been characterized. For example, Cytokines are peptides involved in cell-cell signaling with important physiological effects. They are able to regulate essential cellular functions such as cell division, growth, and death. Some of these factors have known important biological effects, which have been used in the treatment of several diseases.

This specification presents solutions to at least the following historical problems:

1. Selected factors are typically been used as chemicals at artificially created levels that do not simulate levels found naturally in the body. The physiologically imbalanced products are being used to treat disease but have been shown to cause severe, undesired side-effects in clinical settings.

2. No standardization process has been established to create a reproducible product using these secretory factors due to the lack of consistency in the profile.

3. No process in which the profile and/or patterns of signals/factors produced by the cells can be replicated with consistency for scalability.

As disclosed herein, this specification discloses novel solutions to the above presented problems in addition to other issues and problems not listed above. With this platform we are able to collect a set of factors that are chemically and physiologically balanced so that they may be used for therapeutic, medicinal and/or cosmetic purposes. This physiological balance may yield products that have the same or better results, without the undesired effects seen in the current methods. The process disclosed herein also provides a more efficient means of generating differentiated cells, including but not limited to, bone, hair, tissue, cartilage, and almost any other type of cell. The combination of factors, Consortia Factors as detailed herein, may be used for a variety of purposes both in vivo and in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

Various other objects, features and attendant advantages of the present invention will become fully appreciated as the same becomes better understood when considered in conjunction with the accompanying drawings, in which like reference characters designate the same or similar parts throughout the several views, and wherein:

FIG. 1 shows aspects of cultured media, factors and differentiated cells according to exemplary embodiments hereof;

FIGS. 2-8 show aspects of culturing techniques according to exemplary embodiments hereof;

FIG. 9 shows aspects of conditioned media according to exemplary embodiments hereof;

FIGS. 10a-10d show aspects of cell differentiation according to exemplary embodiments hereof;

FIGS. 11a-11e show aspects of secretory factor levels according to exemplary embodiments hereof;

FIG. 12 shows aspects of aspects of factor levels according to exemplary embodiments hereof;

FIGS. 13a-13e show aspects of secretory factor levels according to exemplary embodiments hereof;

FIG. 14 shows aspects of aspects of factor levels according to exemplary embodiments hereof;

FIGS. 15a-15e show aspects of secretory factor levels according to exemplary embodiments hereof;

FIG. 16 shows aspects of aspects of factor levels according to exemplary embodiments hereof;

FIGS. 17a-17e show aspects of cell characteristics according to exemplary embodiments hereof;

FIGS. 18a-18d show aspects of cell characteristics according to exemplary embodiments hereof;

FIGS. 19a-19d show aspects of conditioned media factor profiles according to exemplary embodiments hereof;

FIG. 20 shows a table of factor profiles according to exemplary embodiments hereof;

FIG. 21 shows a table of cell characteristics in conditioned media according to exemplary embodiments hereof; and

FIG. 22 shows a table of factor profiles according to exemplary embodiments hereof.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The following detailed description is not intended to limit the current invention. Alternate embodiments and variations of the subject matter described herein will be apparent to those skilled in the art.

This specification is organized into several main sections to facilitate the clear understanding of the novel subject matter described herein. These sections include:

I. Overview

II. Description of Novel Technologies and Developments

III. Support Information and Data

The Overview section provides an introduction and general overview of the novel technologies and developments described herein.

The Description of Novel Technologies and Developments section provides a detailed description of the novel systems, methods and new technologies that have been developed.

The Support Information and Data section provides detailed research, experimentation and production results that support the novel technologies and developments described herein. This section also includes methodologies that have been developed in accordance with the novel technologies.

I. Overview

Aspects of some exemplary embodiments and methods of the current invention include, without limitation, one or more novel conditioned media, a novel method to formulate and produce the conditioned media, novel factors and combinations of novel factors produced and/or included in the conditioned media, a novel method to produce and/or derive factors from the conditioned media, novel uses of the conditioned media, novel uses of the factors and combinations of factors obtained from the conditioned media, novel uses of the conditioned media and the factors included therein, as well as other aspects of the conditioned media, the factors included in the conditioned media, and other elements and characteristics of the conditioned media.

In some exemplary embodiments hereof, the conditioned media may include a cultured media conditioned with eukaryotic cells, human stem/stromal cells and/or human fibroblasts, animal stem cells, plant stem cells (e.g. meristematic cells), and/or any combination thereof cultured together, individually and/or in any combination. Additional details of the types of cells and/or combinations of cells will be provided in other sections.

The cells may grow in any system, including but not limited to, two and/or three-dimensional scaffolds using any form of suspension, traditionally a liquid/gel suspension and in a controlled static or dynamic system, such as spinner bottles and/or bioreactors. The invention describes the method to culture these cells in a controlled system to facilitate the establishment of effective cell-cell interactions and to collect the conditioned media that contains the various profiles of factors that may be used in vitro or in vivo for uses ranging from cosmetics, to pharmaceuticals, to agriculture.

One aspect of the invention is the method of co-culturing, simultaneously or in successive inoculations, animal cells such as adipose stem cells, derived from different donors, or the same donor but with stem cells taken from different locations on the body or taken at different times or with cells introduced at different passages. For example, when using human cells, the cells may be taken from different parts of the human body such as from the thigh and from the abdomen. In another example, the cells may be taken hours apart up to weeks apart depending on the growth rates and the types of cells. This novel methodology allows for unique cell-cell communication that promotes the secretion of soluble factors mimicking physiologically and chemically balanced levels and profiles. Additionally, the methodology optimizes cell-cell interactions allowing for scalability.

An additional aspect of the invention is the method of co-culturing, simultaneously or in successive inoculations, plant cells such as meristematic stem cells, derived from different plants, or the same plant but with cells taken from different locations on the plant or taken at different times or with cells introduced at different passages. For example, when using plant cells, the cells may be taken from different parts of the plant body such as from the root and from the shoot. In another example, the cells may be taken hours apart up to weeks apart depending on the growth rates and the types of cells. This novel methodology allows for unique cell-cell communication that promotes the secretion of soluble factors mimicking physiologically and chemically balanced levels and profiles. Additionally, the methodology optimizes cell-cell interactions allowing for scalability

In addition to the co-culturing of animal cells with other animal cells, or plant cells with other plant cells, our method also allows for co-culturing, simultaneously or in successive inoculations, of the animal cells with plant cells.

Additionally, the combination of factors released by human stem cells that affect plant meristematic cells and factors released by plant meristematic cells that affect human cells, promotes the production of novel factors with relevant therapeutic effects. A representation of this is depicted in FIG. 1.

In general, it has been determined that the elements within the formulated conditioned media (as described above and herein) interact in a way that one element may regulate the factors secreted by the other element(s).

Our methodology enables not only the concentration of these factors in solution but also the ability to recover the media in sterile conditions suitable for the formulation of pharmaceutical products, cosmetic products, agricultural products and other applications and/or products.

As is known in the art, stem cells may be cultured by adding the cells to a media, often referred to as cultured media, that may include the nutrients necessary to support the cells as they grow and multiply. Culture media compositions may typically include essential amino acids, salts, vitamins, minerals, trace metals, sugars, lipids, nucleosides as well as other components and elements necessary for the cells' growth and development. Cell culture media attempts to supply the components necessary to meet the nutritional needs required to grow cells in a controlled, artificial and in vitro environment. Nutrient formulations, pH, and osmolarity may vary in accordance with parameters such as cell type, cell density, and the culture system employed. Once the culture media is incubated with cells, the culture media may be referred to as “spent” or “conditioned media.

As the cells grow, they may secrete a variety of cellular metabolites and secreted proteins, including, for example, biologically active growth factors, inflammatory mediators and other extracellular proteins. These secretions may be generally referred to as “factors” or “secretomes”. In this way, the conditioned media may contain many of the original components of the media, as well as the secreted factors from the cells.

Much of the research in this field has been focused on the cultured stem cells themselves. The byproduct of the cells and the fluid in which they were cultured/grown (the conditioned media) is usually discarded. Other research has focused on the conditioned media. For example, some research has isolated particular factors in the conditioned media and separated them out. However, the subject matter described within this specification focuses on the cultured media in its totality. Rather than look at individual cell types or individual factors (which traditional methods may immortalize and then use) this technology focuses on the totality of the factors in a physiological balance, in a balance controlled to achieve specific outcomes (i.e. differentiation).

Prior research has discussed using conditioned media from human sourced cells. For example, the conditioned cell media compositions of prior work may be conditioned using any eukaryotic cell type. In some examples, the media may be conditioned by stromal cells, parenchymal cells, mesenchymal stem cells, liver reserve cells, neural stem cells, pancreatic stem cells, and/or embryonic stem cells. This is described in detail in U.S. Pat. No. 6,372,494, the entire contents of which are hereby fully incorporated herein by reference for all purposes.

The new work however, according to exemplary embodiments describe herein, may include conditioned media from plant cells, and/or conditioned media from a combination of plant cells and/or eukaryotic cells (including human and/or animal cells) together to create a new and unique conditioned media. For the purposes of this specification, this new conditioned media is referred to as “Consortia Media”.

This specification describes a novel technology comprising the method of making and using the conditioned media with unique chemical compositions. The conditioned media may include any molecule/compound, including but not limited, to cytokines, peptides, chemokines, and other type of molecule(s)/complex, hereinafter collectively referred to as the “Consortia Factors”. In order to produce Consortia Factors, other cells including but not limited to, eukaryotic cells, animal stem cells and/or fibroblasts and/or plant stem cells alone or in combination, are cultured in any and/or all dimensional constructs or scaffolds.

The Consortia Factors may be produced in different types of defined media and/or Consortia Media being used as culture media. The Consortia Media may be added into any cell culture system. The Consortia Factors may be derived from unique cell-to-cell interactions between animal cells (including human cells), plant cells and any combination thereof. The details of the types of cells and the types of combinations of cells used will be describe in detail in other sections.

The Consortia Factors may be secreted into the media and may be introduced into final products either in a liquid form, or other processed forms, such as powder, emulsion, frozen, etc. The products may include any delivery system, including but not limited to, injection, ingestion, or topical. Example resulting products may include human/animal cosmetics, biopharmaceutical products, nutritional products, agricultural products and applications, bio-energy applications and other products and/or applications.

As described above, the Consortia Media may include factors grown from multiple sources (plant, animal and/or any combination thereof). The growth factors derived from using these cells together, in what is best described as a consortium of factors, is unique. The Consortia Factors may facilitate and increase the expected physiological growth of the cells. The Consortia Factors may also create a uniformity of factor profiles both in content and amount, found in the Conditioned Media, whereas with traditional methods, such consistency may be very limited. In the immediate invention, the constitution of the consortia factors may be very repeatable and predictable across a multitude of similar cultures using similar cells.

Because the constitution, patterns and levels of the Consortia Factors are highly repeatable and standardizable, predefined “formulations” of Consortia Factors may be formed that may be used for different and specific purposes.

For example, in one exemplary embodiment hereof, the Consortia Media and/or Consortia Factors, individually or in any combination, may be produced as “formulations” that may be used to direct cells (in vivo and/or in vitro) to regenerate, differentiate, repair, replace and/or induce very specific desired eukaryotic cells. In this way, the formulations of Consortia Media and/or the Consortia Factors may be used to help bone, cartilage, muscle, skin, tendon, hair, tissue, and/or organs to repair and/or regenerate. This not only enhances the repair/regeneration of damaged cells but may shorten the length of time it takes for such repair/regeneration to occur.

In another exemplary embodiment hereof, the consortia media and consortia factors may also allow for the creation of agricultural products, without having to cultivate any plants.

In another exemplary embodiment hereof, a process has been created through which the conditioned media is reintegrated with fresh cultured media to create a new media in which the stem cells are cultured creating a new set of factors/profile of factors and again creating consistency in these factors and the conditioned media, thus creating Consortia Media. This process may be performed using various methods as described in other sections.

II. Description of Novel Technologies and Developments

In some exemplary embodiments hereof, this invention relates to factor profiles (Consortia Factors) that are released by cells into unique conditioned media (Consortia Media) after cell-to-cell interactions between (without limitation):

-   -   1) Animal cells (including, but not limited to, human cells) and         other animal cells;     -   2) Animal cells and plant cells;     -   3) Plant cells and other plant cells; and/or     -   4) Any combination thereof.

These cells may grow in any culture including two or three-dimensional cultures. The novel methodology allows for the ability to standardize and customize the level and profile of factors released by these cell-to-cell interactions.

A culture media (also referred to as growth media) may include a liquid or gel designed to support the growth of cells. Culture media generally comprise an appropriate source of energy and compounds that may regulate the cell cycle while maintaining pH levels and osmolarity. A typical culture media may include nutrients, essential amino acids, vitamins, inorganic salts, glucose, minerals, serum and other elements necessary to promote the growth of cells in a controlled cultured environment. Once the culture media has been inoculated with the desired cells to be cultured, the combination of cells and culture media may be referred to as a conditioned media.

Cultured cells within a conditioned media may secrete factors that may include heterogeneous groups of proteins that may regulate the growth and differentiation of the cells.

In one exemplary embodiment hereof, a novel conditioned media may be formed by combining a conditioned media of a first type of cells (cells #1) with a conditioned media of a second type of cells (cells #2). Additional conditioned media including additional types of cells (cells #n) may also be added. For the purposes of this specification, the term Consortia Media may refer to the combination of two or more conditioned media. That is, a Consortia Media may consist of two or more distinct conditioned media (e.g., cultures of cells #1, cultures of cells #2, . . . cultures of cells #n) combined together.

Cells #1, cells #2, . . . cells #n may come from one or more sources (source #1, source #2, . . . source #n, respectively). In one exemplary embodiment hereof, the sources #n may be plant, animal and any combination thereof. For example, the consortia media may be formed using the following combinations of cells (without limitation):

-   -   1) Animal cells (cells #1) and other animal cells (cells #2);     -   2) Animal cells (cells #1) and plant cells (cells #2);     -   3) Plant cells (cells #1) and other plant cells (cells #2);         and/or     -   4) Any combination thereof.

Note that an animal source may include a human source. The sources #n may be distinctly different sources and/or the sources #n may be a single source but with samples taken from different areas on the source (e.g., abdomen/thigh for human, roots/shoot for plants, etc.), at different time intervals (e.g., minutes apart, hours apart, days apart, weeks apart, etc.), using other variations and/or any combination thereof.

In another embodiment hereof, a novel conditioned media may be formed by co-culturing a first type of cells (cells #1) with a second type of cells (cells #2). Additional types of cells (cells #n) may also be added. For the purposes of this specification, in addition to the combination of two or more conditioned media, the term Consortia Media may refer to the conditioned media resulting from the aforementioned co-culturing of different types of cells.

For the purposes of this specification, conditioned media #1 may include culture media #1 inoculated with cells #1 taken from a source (e.g., from source #1), conditioned media #2 may include culture media #2 inoculated with cells #2 taken from a source (e.g., from source #2 or from a variation of source #1), and conditioned media #n may include culture media #n inoculated with cells #n taken from a source (e.g., from source #n or from a variation of other sources used). Note that any Source #n may be the same as any other Source #n, but taken at different times, from a different location on the source, using any other variation or any combination thereof. Alternatively, any Source #n may be a distinctly different source than any other Source #n.

Cells #1, #2, . . . #n may include without limitation:

-   -   1) Eukaryotic cells, animal (including but not limited to human,         other mammals, avian, rodent, etc.) stem/stromal cells and/or         animal fibroblasts, endothelial cells, Induced Pluripotent Stem         cells (IPS cells) from different sources (whether from different         places in a single animal donor's body, from a single animal         donor taken from the same location but at a different date of         sample collection, from multiple sources or from multiple animal         donor bodies) growing in specific serum-free or serum-based         culture/induction/differentiation media.         -   For example, co-culturing, simultaneously or in successive             inoculations between, without limitation:         -   i. Animal cell & same Animal cell from successive             inoculation times;         -   ii. Animal cell & same Animal cell from different locations             on the animal from simultaneous inoculations;         -   iii. Animal cell & different Animal cell from simultaneous             inoculation;         -   iv. Animal cell & different Animal cell from successive             inoculations;     -   2) Different types of plant stem/meristematic cells from the         initial plant embryonic stem cells (totipotent) in shoot apical         meristem and root apical meristem (SAM and RAM respectively) and         the plant stem/meristematic cells derived from the multipotent         stem cells such as vascular cambium, individually or derived         from multiple types of plant meristematic cells.         -   For example, between, without limitation:         -   i. Plant stem cell of a genus & plant stem cell of the same             genus of successive inoculation times;         -   ii. Plant stem cell of a genus & plant stem cell of the same             genus of simultaneous inoculation;         -   iii. Plant stem cell of genus 1 & plant stem cell of genus             of #n simultaneous inoculation;         -   iv. Plant stem cell of genus 1 & plant stem cell of genus #n             of successive inoculations.     -   3) Eukaryotic cells, animal stem/stromal cells and/or animal         fibroblasts, endothelial cells, iPS cells (either from an         individual donor or from multiple donors) and/or plant         meristematic cells.     -   4) Eukaryotic cells, animal stem/stromal cells and/or animal         fibroblasts, endothelial cells, and/or iPS cells that grow in         the conditioned media derived from plant meristematic cells;     -   5) Plant meristematic cells that grow in the conditioned media         derived from Eukaryotic cells, animal/human stem/stromal cells         and/or animal/human fibroblasts, endothelial cells, and/or IPS         cells.     -   6) Plant cells introduced into animal cultured media;     -   7) Other types or combinations of types of cells.     -   8) Co-culture(s) of any combinations derived from using one or         more of the above cells and/or specific         culture/induction/differentiation media.     -   9) Creating a system in which all of the above interact, and         stimulate cell to cell interactions between the various cultures         being grown, as describe in further detail herein below, so as         to stimulate growth of new and additional growth factors         creating a unique culture media that is the Consortia Media         described herein.     -   10) The cell-cell interactions described herein above may occur         through direct contact between the cells or using the culture         media as a conduit for the cell-cell interactions.

In general, the process starts with cells being cultured in culture media. This culturing of cells can be done in one sequential co-culturing in the same receptacle, or in different receptacles and then integrated. The conditioned media from one set of cultured stem cells may be integrated with the conditioned media from other cultured cells (e.g., in the various combinations described above in 1-10). This combination may be performed in different ways for different results/types of Consortia Factors, including but not limited to: unilaterally in a directional flow; bidirectional Serial System, Bidirectional Loop System; Additive Uni/Bi-directional System; and, cell co-culturing to create the Consortia Media.

In one exemplary embodiment hereof, the process of creating consortia media and consortia factors may begin by taking cells of diverse sources and culturing them. The source of the cells may be taken from a multitude of scenarios, and the diversity of cell sources may facilitate the creation of Consortia Media and Consortia Factors. The diversity may come from harvesting different types of cells and conditioned media as described above.

In another embodiment of the technology, the interaction of conditioned media #1, conditioned media #2, . . . conditioned media #n may be combined and/or formed in any manner of combination, including but not limited to the following scenarios: sequential co-culturing in the same receptacle (FIG. 2), sequential unidirectional system (FIG. 3), bidirectional serial system (FIG. 4), cell co-culture (FIG. 5), unidirectional loop system (FIG. 6), bidirectional loop system (FIG. 7), and/or additive uni/bi-directional system (FIG. 8). This interaction between the different conditioned media creates new forms of conditioned media with new factor profiles. This combined media and the resulting factors/factor profiles are Consortia Media and Consortia factors respectively.

In one example, cells #1 may be cultured in a culture media #1 to form a conditioned media #1, and cells #2 may be cultured in a culture media #2 to form a conditioned media #2. Then, the conditioned media #1 and the conditioned media #2 may be added together to form the overall conditioned media (the “Consortia Media”)

In another example, cells #1 and cells #2 may be cultured in the same culture media (simultaneously or sequentially) to form the overall conditioned media (the “Consortia Media”)

In addition, the media composition for the cell cultures may include, without limitation:

-   -   1) Cell culture media with serum;     -   2) Serum-free cell culture media;     -   3) Conditioned media as an additive to cell culture media;     -   4) Cultured media as cell culture media;     -   5) Consortia Media as an additive to cell culture media;     -   6) Consortia Media as cell culture media; and     -   7) Any other types of cell culture elements and/or any         combination thereof.

In one exemplary embodiment hereof, once the consortia media is formed, the cells within the consortia media (cells #1, cells #2, . . . cells #n from possible source #1, source #2, . . . source #n) may interact with one another and secrete novel factor profiles. For the purpose of this specification, the novel factor profiles secreted by the cells within the consortia media may be referred to as Consortia Factors. It is understood that two or more consortia media may be combined to form additional tiers of combined consortia media that may produce additional types of Consortia Factors. Further details and examples of this are described in Section III of this specification.

The consortia factors may include any molecule and/or compound, including but not limited to, cytokines, peptides, chemokines, and other type(s) of molecule(s) and/or complex(es) and any combination thereof. In order to produce consortia factors, animal stem cells (including human stem cells and/or stem cells from any known animal or being) and/or fibroblasts and/or plant stem cells alone or in combination may be cultured in any and/or all dimensional constructs or scaffold suspended in different types of defined and/or culture media (e.g. consortia media) added into cell cultures and/or bioreactors in a controlled growth cultured system, whether that system is a closed or open system.

Such systems create a platform in which any or all of the above interact. The interaction creates Consortia Media to be reintegrated into the process described or to create an interaction between them so as to stimulate growth of new and additional Consortia Factors. This in turn creates a new and unique Consortia Media different from those described herein above. The Consortia Media/Factors can also be used as a final product to be introduced into various other products for use/sale in the marketplace.

Traditionally, stem cells and/or the factors they produce, have been cultured with the goal of creating a specific “legacy” lines of cells or factors that may be reproduced with the goals that the specifically identified legacy cells/factors may treat specific health ailments or problems. The technology described herein fundamentally differs from that concept in a number of ways.

First, rather than looking for a legacy line of cells, the technology described herein uses the media in which the cells were cultured. In doing so, a “holistic” approach towards the factors is created that is more physiologically balanced and may thereby stimulate cells more effectively resulting in safer healing and regeneration.

Second, rather than looking at keeping a legacy line alive, the technology described herein creates effective cell-to-cell interactions between various types of cells as described herein, allowing for the factor profiles (peptides, non-protein growth factors, vitamins, fatty acids, extra nucleic acids, lipids, cytokines, small molecules, etc.) aimed at a desired differentiation to be standardized.

Third, the technology described herein includes using a combination of plant meristematic cells and/or their conditioned media to create new factor profiles. Additionally, combination of plant meristematic cells with animal cells and/or their conditioned media can be used to create new factor profiles.

In one exemplary embodiment hereof, the cell-cell interactions as described herein may be optimized in a way to obtain combinatorial effects from the factors found in the conditioned media that may not exist otherwise. These cell-cell interactions may include cells at different culture stages and origins such as plant meristematic cells that grow with other meristematic cells from the same or different plant, and/or animal or human cells that grow with cell co-culturing to create the Consortia Factors and Consortia Media. Unlike prior uses of factors or media, the co-culturing that may create the Consortia Factors and Consortia Media demonstrates unique characteristics such as stability, consistency, repeatability in forming the desired Consortia Factors and desirable cellular effects unseen in prior art.

Co-culturing of different types of cells or of cells in different stages of differentiation has not been described, until now, in terms of their secretory products. It is this unique product that can be used in vivo or in vitro to direct a specifically desired effect on the cells.

The Consortia Media and/or the Consortia Factors, individually or in any combination, may be used in a wide range of applications including health products, biopharmaceutical products, personal care products, agricultural products and other products.

In one exemplary embodiment hereof, because the Consortia Media and/or Consortia Factors may be reproduced so efficiently, specific sets of “formulations” of media may be used to cultivate the stem cells, resulting in specific and consistent sets of factor profiles released. After cultivation, these formulations may contain standardized levels of biologically active secretory factors (Consortia Factors). These consistent sets of factors may then be used to increase the efficacy (in time and potency) of the differentiation.

The standardization of secretory factor levels may be quantified through a multitude of successive cell inoculations as described above, and then by quantifying the factor levels and patterns within each inoculation. That is, different types of Consortia Media may be used to form different types of Consortia Factors, and then the Consortia Media and Consortia Factors may be analyzed to determine their characteristics and compositions. Experimentation may continue until a desired Consortia Media and/or Consortia Factor is achieved, and then, since the results of this new technology of forming the media and/or the factors is so repeatable, standardization may be achieved. These cells may be inoculated into spinner bottles or bioreactors. This methodology alters the variations of the secretory factor profiles produced by the cells. It has been determined that this process is reproducible as is demonstrated with the embodiments referenced in FIG. 9. The descriptions and methodologies are described in detail in Section III of this specification.

The differentiations may occur either in vitro, or when introduced in vivo into the host (note that the host can be human, other animals, agriculture and/or any combination thereof). Each formula may be designed to stimulate a specific type of differentiation. For example, the formula for the media that may produce bone when applied to a patient may be different than the formula for the media that may produce hair. With these specific formulas applied to patients with specific needs, the recovery time for particular types of injuries may be greatly reduced. For example, the recovery time from broken bones may be reduced from months to weeks. This may also be used for other types of regenerative medicine ranging from organs to hair growth.

In further exemplary embodiments hereof, the Consortia Media and/or Consortia Factors, individually or in any combination, may be used to treat conditions such as dry eyes, psoriasis, eczema, auto immune diseases, alopecia and other conditions.

Since the differentiated cells may be reduced to liquid form, powder and/or gas, the delivery method of products that may include the Consortia Media and/or the Consortia Factors for health or other types of treatments may include injection, inhalation, topical, ophthalmic, otic, and/or ingestion as a distinct treatment and/or in any combination with other treatments. The Consortia Media and/or Consortia Factors, alone or in any combination, may be introduced into final products in liquid form, solid (frozen media, powder), gases/inhalants (e.g., micro-dispersion, dry powder inhaler, aerosol), other processed formats, other forms and any combination thereof.

The Consortia Media and/or Consortia Factors, individually or in any combination, may also be used to treat similar conditions in other animals in addition to humans (e.g., dogs, cats, horses, etc.) by using similar methods as described.

In one exemplary embodiment hereof, the Consortia Media and/or Consortia Factors, individually or in any combination, may be used in agricultural applications. In one exemplary application, the Consortia Media and/or Consortia Factors may be used to cultivate and produce plant products without the need to cultivate the plants themselves. The benefits of this are multifold. First, this may allow for the ability to create food grade plant products using only a fraction of natural resources typically needed such as water, soil, nutrients and other resources. The food grade products may also be formed without the need for herbicides, pesticides, or other harmful components. In addition, the chemical and mineral content of the resulting products may be controlled, designed and/or manipulated.

In one example, plant meristematic cells may be used to differentiate the cells into any type of cell desired, and as such, the cells may be guided to grow into the cells that constitute a specific plant product. For example, it is known that oranges may be high in vitamin C. Traditionally, to get vitamin C from an orange, an entire orange tree must be grown that may provide the orange fruit. This process requires a great deal of water, soil and nutrients to grow the roots, trunk, leaves, branches and the orange. However, using the technology described herein, the cells that constitute the orange may be formed (differentiated) without the need for growing the tree. Note that the actual orange fruit may not necessarily be formed during the cellular differentiation, but instead, the cells that constitute the orange may be formed instead. Experimental examples of this are described in Section III of this specification with regards to flax and its health benefits. As described, the factors in flax which include the health benefits may be produced using this technology without growing the entire flax plant.

In another example, eucalyptus leave meristematic cells may be cultured and differentiated into the equivalent of the leaves. The essential oils and elements from those “leaf cells” may then be used for the health benefits they may provide, all without having grown a eucalyptus tree, which is notorious for the large amount of water it may take.

Additionally, the Consortia Factors may be grown with a consistent profile not subject to all the other influences traditional agriculture may be subject to. For example, grapes used for wine may have a different profile each year they are grown depending on the location, weather, soil, etc., all of which create inconsistencies within the grapes. This may be why a specific wine may have a distinctive and different taste from year to year, even though grown from the same seeds, in the same vineyard, with the same methodologies. However, by growing the cells, rather than the entire grape, these variables may be controlled to create a more uniform bouquet to the wine. Additionally, harmful pesticides, herbicides or other chemicals may be avoided.

In another example, the levels of vitamin “C” found in oranges may be increased. In another example, plants that may not typically include vitamin “C” may be grown such that they do. It can be seen that the growth of plant products without cultivation may also allow for growth of food products in environments that may not otherwise grow these foods.

As an additional benefit of the ability to cultivate plant materials without actual cultivation, this technology may induce photosynthesis, with minimal light, water, gases or other natural resources.

It is understood by a person of ordinary skill in the art, upon reading this specification, that any plant substance may be formed in this way, and that the scope of the invention is not limited in any way by the types of plant substances, media and/or factors that may be formed. For example, it is contemplated that the growth of substances like THC and CBD may be provided without the need for growing the actual plants. In this way, what may currently take acres of land to cultivate cannabis plants, may be done in a laboratory setting instead.

In addition to using the Consortia Media (and general conditioned media) for these products rather than discard it, the biomass from the media may include specific plant products that may allow the material to be formed as a food source and/or dietary supplement. Such food sources and/or dietary supplements may then be used in applications such as terraforming and space travel. The biomass may also be used for bio-diesel products and/or agricultural products (again with beneficial implications for terraforming and space travel). It is understood that not only may plant products be produced, but what would normally be considered waste in the industry, may also be formed into beneficial products.

For agricultural applications, the Consortia Media and/or the Consortia Factors may be delivered as additives to soil, as genetically engineered/modified plants or by other means.

It is understood by a person of ordinary skill in the art that the above examples are meant for demonstration and that the systems and methods described herein may be used to orchestrate other benefits within the fields of health, biotech, personal care, agriculture, cosmetics and other fields and applications, and that the scope of the systems and/or methods are not limited in any way by the fields into which they may be applied or by the benefits that they may provide.

III. Support Information and Data

Multiple experiments were performed, as detailed below, using two different methods when co-culturing cells. One without direct cell-cell contact and the other with direct cell-cell contact (FIGS. 4 and 5 respectively). The two resulting Consortia Media derived from these interactions were analyzed by cytokine multiplex analysis and compared to controls. (FIG. 9 and FIG. 20 (TABLE 1)). The data demonstrates novel secretary factor profiles compared to controls. The pattern also demonstrated attenuated profiles of pro-inflammatory factors and chemokines. This has direct implications for pharmacological products.

Conditioned Media Vs. Consortia Media Experiments

Adipose stem cells from the same donor were expanded and plated at high density in serum-free media. Chemically defined media was added to these cell cultures to promote differentiation to bone, cartilage and fat. After 20 days in culture, the cells were tested for differentiation. The remaining conditioned media recovered from the differentiation experiment was then used as culture media on ASCs. After 20 days in culture, the cells contained in the newly created Consortia Media were tested for differentiation. The profiles from the conditioned media and the Consortia Media were then analyzed. The results from each experiment are detailed below.

Conditioned Media from Defined Osteogenic Media Vs. Osteogenic Consortia Media from Conditioned Media

FIGS. 11a-11e show graphic representations and statistical analysis of one exemplary embodiment of the technology. The figures show secretory factor levels during differentiation of ASCs to bone, with 11(a) ASCs growing in SFM, 11(b) ASCs treated with growth factors (GF) to induce bone differentiation, 11(c) ASCs treated with Osteogenic Conditioned Media to induce bone differentiation, 11(d) Statistical analysis showing significant differences between the level of factors from ASCs growing in SFM vs ASCs treated with growth factors, and 11(e) Statistical analysis showing significant differences between the level of factors from conditioned media of ASCs growing in SFM vs. Consortia Media of ASCs treated with Osteogenic Conditioned Media. The results are taken from three independent experiments using Pairwise t-tests (P value).

FIG. 12 factor profiles of ASC's treated with Defined Osteogenic Media versus Osteogenic Conditioned Media, demonstrates one exemplary embodiment of the novel technology. In this exemplary embodiment, the data demonstrates that this novel approach provides a consistent, reproducible set of factors for differentiation into bone. This Osteogenic Consortia Media shows an enhancement of the differentiation process (FIGS. 10a, 10d ).

Conditioned Media from Defined Chondrogenic Media Vs. Chondrogenic Consortia Media from Conditioned Media

FIGS. 13a-13e show graphic representations and statistical analysis of one exemplary embodiment of the technology. The figures show secretory factor levels during differentiation of ASCs to cartilage, with 13(a) ASCs growing in SFM, 13(b) ASCs treated with growth factors (GF) to induce cartilage differentiation, 13(c) ASCs treated with Chondrogenic Consortia Media to induce cartilage differentiation, 13(d) Statistical analysis showing significant differences between the level of factors from ASCs growing in SFM vs ASCs treated with growth factors, and 13(e) Statistical analysis showing significant differences between the level of factors from conditioned media of ASCs growing in SFM vs. Consortia Media of ASCs treated with Chondrogenic Conditioned Media. The results are taken from three independent experiments using Pairwise t-tests (P value).

FIG. 14 shows Factor Profiles of ASC's treated with Defined Chondrogenic Media versus Chondrogenic Conditioned Media, demonstrates one exemplary embodiment of the novel technology. In this exemplary embodiment, the data demonstrates that this novel approach provides a consistent, reproducible set of factors for differentiation into cartilage. This Chondrogenic Consortia Media shows an enhancement of the differentiation process (FIGS. 10a, 10c ).

Conditioned Media from Defined Adipogenic Media Vs. Adipogenic Consortia Media from Conditioned Media

FIGS. 15a-15e show graphic representations and statistical analysis of one exemplary embodiment of the technology. The figures show secretory factor levels during differentiation of ASCs to fat, with 15(a) ASCs growing in SFM, 15(b) ASCs treated with growth factors (GF) to induce fat differentiation, 15(c) ASCs treated with Adipogenic Consortia Media to induce fat differentiation, 15(d) Statistical analysis showing significant differences between the level of factors from ASCs growing in SFM vs ASCs treated with growth factors, and 15(e) Statistical analysis showing significant differences between the level of factors from conditioned media of ASCs growing in SFM vs. Consortia Media of ASCs treated with Adipogenic Conditioned Media. The results are taken from three independent experiments using Pairwise t-tests (P value).

FIG. 16 shows Factor Profiles of ASC's treated with Defined Adipogenic Media versus Adipogenic Conditioned Media, demonstrates one exemplary embodiment of the novel technology. In this exemplary embodiment, the data demonstrates that this novel approach provides a consistent, reproducible set of factors for differentiation into fat. This Adipogenic Consortia Media shows an enhancement of the differentiation process (FIGS. 10a, 10b ).

Plant Cell Cultures and their Combination with Other Plant or Animal Cells

In general plant cell cultures are developed with the plant tissue in culture media with plant growth hormones that de-differentiate plant cuttings, containing differentiated cell types/tissues, to form a totipotent cell mass called a callus which is a meristematic cells. These meristematic cells form the entire plant when grown in growth hormones that can differentiate them to shoot and root systems. Plant cell culture lines are established with plant cells in liquid culture.

In the present exemplary embodiment of this process, two different plant species were co-cultured in the same media, cell #1 and cell #2. The combination two different cells being co-cultured at once, created the Consortia Media. Every 24 hours, for 8 days, samples of the Consortia Media were harvested and analyzed for cell density and total protein concentration.

When compared to cell cultures of cell #1 and cell #2 cultured separately, higher rates of cell density, the growth of cell culture biomass and protein concentration was found in the Consortia Media as demonstrated in FIGS. 17a -17 c.

FIGS. 18a-b and 18c-d (Images at 10× and 20× magnification) show ASCs cultured in 6-well plates precoated with fibronectin in SFM+10% HFCM. After 48 hours, the media was replaced for fresh SFBM-112818ACFSF+10% HFCM-012318ACFSF (Human Media—controls) in 3 wells (#1-3) and 2,4-D/KIN media (Plant Media—LMS) in the rest of wells (#4-6). After 72 hours, pictures of the cells were taken before trypsinization. The viability of growing animal cells in plant conditioned media was confirmed as shown in FIG. 21 (TABLE 2).

Human Adipose Stem Cell & Plant Stem Cell Culture in Human Culture Media

Adipose Stem/Stromal Cell Isolation and Culture:

Same protocol described before for ASC isolation, culture, and expansion.

Adipose Stem Cell Inoculation into Static Bottles with Consortia Media from Flax:

Same protocol described before for cell inoculation into spinner bottles or bioreactor that contains Fibra-cel disks. In this case Hair differentiation serum-free Consortia Media developed in our laboratory is used for spinner bottles or bioreactor. Adipose Stem Cells were maintained undifferentiated in Serum-free Consortia Media developed in our laboratory.

Adipose Stem/Stromal Cell Inoculation into Static Bottles with Flax Biomass:

Flax biomass was growing in Serum-free Consortia Media developed in our laboratory and adipose stem cells were added at a density of 1×10⁵ cells/mL. Then, the consortia media (CSM) was collected after 72 hrs. of cell-cell interactions and evaluated by cytokine multiplex analysis.

Adipose Stem/Stromal Cell Inoculation into Static Bottles with Flax Biomass:

Consortia media from Flax was adding to supplement the Serum-free Consortia Media developed in our laboratory. Then, ASCs were added plated at a cell density of 1×10⁵ cells/mL. The consortia media (CSM) was collected after 72 hrs. of cell-cell interactions and evaluated by cytokine multiplex analysis.

FIGS. 19a-19d show graphic representations of Consortia Media factor profiles. These secretory factor levels were obtained during cell-cell interactions between ASCs and Flax consortia media (Flax CSM) or Flax Biomass (Flax BM) with 19(a) Secretory factor profile of Flax CM or Flax BM using human antibodies that cross react with plant secretory factors, 19(b) ASCs growing in SFM, 19(c) ASCs co-cultured with Flax BM in human culture media, and 19(d) ASCs culture with Flax CM (1%) in human culture media.

FIG. 22 (TABLE 3) shows Cytokine multiplex analysis of Consortia media (CSM) obtained from the interactions between human adipose stem and Flax Biomass (Flax-B) or Flax Consortia Media (FlaxCSM). The values correspond to the cytokine level in pg/mL.

These experiments demonstrate the viability of culturing species in cultured media of other species, thus creating new platforms from which to create novel factor profiles as supported by the previously discussed experiments.

Benefits

Benefits of the exemplary embodiments described herein may include, but are not limited to:

In producing and/or using the Consortia Meida/Factors as described above, we have designed a way to have a consistency of factors being produced. This includes the standardization of the processes necessary to formulate treatments for various diseases/conditions, including but not limited to, human, other animals, plants, and agriculture.

In addition, there have been safety and regulatory concerns (both domestically and internationally) on the use of human derived stem cells. The Consortia Factors, including the plant derived Consortia Factors can generate what in effect are the same type of factors (such as polypeptide factors) obtained from human stem cells. The idea is that the factors generated from the Consortia Factors are more efficient at helping consistent differentiation of the stem cells, including into human cells.

By using the Consortia Factors, we are able to use universally ubiquitous sets of physiologically balanced compounds that will allow the recipient's own body to use the compound in a self-limiting manner to self-regenerate/renew/heal tissues/bone/organs. (This is analogous to giving the body a calcium supplement. The source of the supplement, whether derived from milk, plants, or found as an element in the earth, is irrelevant. What is relevant is giving the patient the calcium. Here what is relevant is not the cell source, but the derivatives i.e. the consortia factors, that produces the desired results).

Agriculture takes tremendous amounts of resources. From land use, to water use, nutrients etc. The process we have developed, particularly with the Consortia Factors enables the growth of agriculture with a fraction of the resources. This is accomplished because we are not actually growing plants, just all the nutrients and byproducts of plants. We can, in essence, grow any plant we want, without actually growing the actual plant. Instead we grow what would be described as a biomass that has all the desired yield of the plant we are not growing. This can result in a food sources, nutritional supplements, medicinal products, Consortia Factors, biofuel, and agricultural products.

The current methodology of deriving stem cells, factors, or Conditioned Media, creates scenarios wherein the profile of the factors and/or cells being produced, are inconsistent, and often unhealthy or have a very short span in which they can be produced or used. In contrast, we are collecting sets of factors from cells that are synchronized and healthy which harmonize the factors, increasing efficacy. Specifically, it will have increased specific activities of factors, such as concentration, types factors, profile of factors, etc. with reasonable consistency (higher than currently attainable).

The conditioned media derived from the cell-cell interactions between human and plan stem cells contains secretory factors that can be used in Regenerative Medicine or Cosmetics.

Cosmetic Applications

The secretory factors are molecules that promote anti-aging effects when applied topically to the skin. Some of these effects are antioxidant and can improve the physical appearance of the skin. Factors such as fibroblast growth factors (FGFs) that play a central role for regeneration of a wide variety of tissues including skin by promoting cellular proliferation and differentiation. Other factors that correspond to small molecules such as interferons (IFNa2, IFNγ) are essential for cellular proliferation and they modulate immune responses. In addition, factors such as Eotaxin-1, granulocyte colony-stimulating factor (G-CSF), macrophage colony stimulating factors (M-CSFs), regulated upon activation normal T cell express sequence (RANTES), epithelial neutrophil activating peptide 78 (ENA-78/CXCL5) re-enforced the immunological system and in this way maintain the integrity of the skin. Interleukins correspond to a group of factors that play an essential role to regulate immune and inflammatory responses in the skin and other tissues. Vascular endothelial growth factor (VEGF) in harmony with some of the factors described above, is involved in maintenance of skin function and regeneration as it occurs during wound healing.

These systems may also work to promote hair differentiation (which will in turn lead to hair growth). In using the Consortia Factors (delivered either topically or injected into the scalp) the natural growth of hair may be stimulated. In this application, Consortia Factors, delivered directly or using them in the Conditioned Media they may be produced in, may help trigger cell/tissue/organ regeneration. This may be true for growth in eukaryotic cells, human cells, animal cells, and even plant cells.

Biopharmaceutical Applications

Some effects exerted by these factors may include but are not limited to the promotion of wound healing, angiogenesis, cellular proliferation, cellular survival, anti-aging effects, antioxidant effects, anti-inflammatory effects, osteogenesis, chondrogenesis, adipogenesis, hair regeneration. These factors can also be beneficial for the treatment of auto-immune diseases and for tissue repair after trauma.

Where a process is described herein, those of ordinary skill in the art will appreciate that the process may operate without any user intervention. In another embodiment, the process includes some human intervention (e.g., a step is performed by or with the assistance of a human).

As used herein, including in the claims, the phrase “at least some” means “one or more,” and includes the case of only one. Thus, e.g., the phrase “at least some ABCs” means “one or more ABCs”, and includes the case of only one ABC.

As used herein, including in the claims, term “at least one” should be understood as meaning “one or more”, and therefore includes both embodiments that include one or multiple components. Furthermore, dependent claims that refer to independent claims that describe features with “at least one” have the same meaning, both when the feature is referred to as “the” and “the at least one”.

As used in this description, the term “portion” means some or all. So, for example, “A portion of X” may include some of “X” or all of “X”. In the context of a conversation, the term “portion” means some or all of the conversation.

As used herein, including in the claims, the phrase “using” means “using at least,” and is not exclusive. Thus, e.g., the phrase “using X” means “using at least X.” Unless specifically stated by use of the word “only”, the phrase “using X” does not mean “using only X.”

As used herein, including in the claims, the phrase “based on” means “based in part on” or “based, at least in part, on,” and is not exclusive. Thus, e.g., the phrase “based on factor X” means “based in part on factor X” or “based, at least in part, on factor X.” Unless specifically stated by use of the word “only”, the phrase “based on X” does not mean “based only on X.”

In general, as used herein, including in the claims, unless the word “only” is specifically used in a phrase, it should not be read into that phrase.

As used herein, including in the claims, the phrase “distinct” means “at least partially distinct.” Unless specifically stated, distinct does not mean fully distinct. Thus, e.g., the phrase, “X is distinct from Y” means that “X is at least partially distinct from Y,” and does not mean that “X is fully distinct from Y.” Thus, as used herein, including in the claims, the phrase “X is distinct from Y” means that X differs from Y in at least some way.

It should be appreciated that the words “first” and “second” in the description and claims are used to distinguish or identify, and not to show a serial or numerical limitation. Similarly, the use of letter or numerical labels (such as “(a)”, “(b)”, and the like) are used to help distinguish and/or identify, and not to show any serial or numerical limitation or ordering.

As used herein, including in the claims, the terms “multiple” and “plurality” mean “two or more,” and include the case of “two.” Thus, e.g., the phrase “multiple ABCs,” means “two or more ABCs,” and includes “two ABCs.” Similarly, e.g., the phrase “multiple PQRs,” means “two or more PQRs,” and includes “two PQRs.”

The present invention also covers the exact terms, features, values and ranges, etc. in case these terms, features, values and ranges etc. are used in conjunction with terms such as about, around, generally, substantially, essentially, at least etc. (i.e., “about 3” shall also cover exactly 3 or “substantially constant” shall also cover exactly constant).

As used herein, including in the claims, singular forms of terms are to be construed as also including the plural form and vice versa, unless the context indicates otherwise. Thus, it should be noted that as used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

Throughout the description and claims, the terms “comprise”, “including”, “having”, and “contain” and their variations should be understood as meaning “including but not limited to” and are not intended to exclude other components unless specifically so stated.

It will be appreciated that variations to the embodiments of the invention can be made while still falling within the scope of the invention. Alternative features serving the same, equivalent or similar purpose can replace features disclosed in the specification, unless stated otherwise. Thus, unless stated otherwise, each feature disclosed represents one example of a generic series of equivalent or similar features.

The present invention also covers the exact terms, features, values and ranges, etc. in case these terms, features, values and ranges etc. are used in conjunction with terms such as about, around, generally, substantially, essentially, at least etc. (i.e., “about 3” shall also cover exactly 3 or “substantially constant” shall also cover exactly constant).

Use of exemplary language, such as “for instance”, “such as”, “for example” (“e.g.,”) and the like, is merely intended to better illustrate the invention and does not indicate a limitation on the scope of the invention unless specifically so claimed.

While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiments, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. 

I claim:
 1. A method for producing factors, the method comprising: (A) providing a first culture media; (B) inoculating the first culture media with first cells to form a first conditioned media; (C) providing a second culture media; (D) inoculating the second culture media with second cells to form a second conditioned media; (E) combining the first conditioned media and the second conditioned media to form a first combined conditioned media; and (F) producing factors in the first combined conditioned media.
 2. The method of claim 1 wherein the first cells include cells taken from a first animal and the second cells include cells taken from a second animal.
 3. The method of claim 2 wherein the first animal and the second animal are the same animal, wherein the first cells include cells taken at a first time and the second cells include cells taken at a second time, and wherein the first time and the second time are different times.
 4. The method of claim 2 wherein the first animal and the second animal are the same animal, wherein the first cells include cells taken from a first region on the animal's body and the second cells include cells taken from a second region on the animal's body, and wherein the first region and the second region are different regions.
 5. The method of claim 2 wherein the first animal and the second animal are different animals.
 6. The method of claim 2 wherein the first animal cells and the second animal cells are selected from the group: eukaryotic cells, stem cells, fibroblast cells and stromal cells.
 7. The method of claim 1 wherein the first cells include cells taken from an animal and the second cells include cells taken from a plant.
 8. The method of claim 1 wherein the first cells include cells taken from a first plant and the second cells include cells taken from a second plant.
 9. The method of claim 8 wherein the first plant and the second plant are the same plant, wherein the first cells include cells taken at a first time and the second cells include cells taken at a second time, and wherein the first time and the second time are different times.
 10. The method of claim 8 wherein the first plant and the second plant are the same plant, wherein the first cells include cells taken from a first region on the plant's body and the second cells include cells taken from a second region on the plant's body, and wherein the first region and the second region are different regions.
 11. The method of claim 8 wherein the first plant and the second plant are different plants.
 12. The method of claim 1 wherein the growth factors include cytokines, chemokines and/or small molecules.
 13. The method of claim 1 further comprising the step: (G) using the first combined conditioned media formed in (E) and/or the factors produced in (F) to promote cellular differentiation.
 14. The method of claim 1 further comprising the step: (H) using the first combined conditioned media formed in (E) and/or the factors produced in (F) to regenerate bone, cartilage, muscle, skin, tendon, hair, tissue and/or organs.
 15. The method of claim 13 wherein the cellular differentiation includes animal cellular differentiation.
 16. The method of claim 13 wherein the cellular differentiation includes plant cellular differentiation.
 17. The method of claim 16 wherein the plant cellular differentiation forms plant material.
 18. The method of claim 17 wherein the plant material induces photosynthesis.
 19. The method of claim 18 wherein the plant material is used for terraforming.
 20. The method of claim 1 further comprising the step: (I) using the first combined conditioned media formed in (E) and/or the factors produced in (F) in pharmaceutical, medical, cosmetic and/or agricultural products. 